Changes of small bowel motility and noradrenaline content of the intestinal wall in response to alpha- and beta-adrenergic blockade in dog

In experiments on dogs, the spontaneous movements of the small bowel were in all cases enhanced by the alpha 2-blocker phentolamine, while they were not influenced, or were slightly decreased, by the beta 1-blocker practolol. Neither drug caused a change in the noradrenaline content of the intestinal wall. In the same animals, the joint administration of phentolamine and practolol led to a considerable increased small bowel motility, and to a significant decrease in the noradrenaline level of the intestinal wall. The results are in agreement with experimental data indicating that presynaptic alpha-receptors play a primary role in the sympathetic regulation of small bowel motility.     

Distribution of the kinin-breakdown activity in areas of the rat brain in the dynamics of spontaneous hypertension

Kinin-damaging activity (KDA) has been studied in 8 brain areas of normotensive rats and rats with spontaneous hypertension, aged 3 to 12 months. A significant depression in KDA was revealed in midbrain, striatum, thalamus, and pituitary body of normotensive rats 6 months of age, as compared to 3-month-old animals. A tendency towards KDA increase was noted in the hypothalamus. In 12-month-old normotensive rats KDA level returned to baseline (3 months of age). Comparison of KDA in rats with spontaneous hypertension aged 3 to 12 months has revealed no age-dependent differences and it is, therefore, believed that rats with spontaneous hypertension lack certain mechanisms inducing a considerable decrease of KDA in 6-month-old normotensive rats.     

DNA binding properties of human Cdc45 suggest a function as molecular wedge for DNA unwinding

The cell division cycle protein 45 (Cdc45) represents an essential replication factor that, together with the Mcm2-7 complex and the four subunits of GINS, forms the replicative DNA helicase in eukaryotes. Recombinant human Cdc45 (hCdc45) was structurally characterized and its DNA-binding properties were determined. Synchrotron radiation circular dichroism spectroscopy, dynamic light scattering, small-angle X-ray scattering and atomic force microscopy revealed that hCdc45 exists as an alpha-helical monomer and possesses a structure similar to its bacterial homolog RecJ. hCdc45 bound long (113-mer or 80-mer) single-stranded DNA fragments with a higher affinity than shorter ones (34-mer). hCdc45 displayed a preference for 3′ protruding strands and bound tightly to single-strand/double-strand DNA junctions, such as those presented by Y-shaped DNA, bubbles and displacement loops, all of which appear transiently during the initiation of DNA replication. Collectively, our findings suggest that hCdc45 not only binds to but also slides on DNA with a 3′–5′ polarity and, thereby acts as a molecular ‘wedge’ to initiate DNA strand displacement.     

Metabolism of carbosulfan II. Human interindividual variability in its in vitro hepatic biotransformation and the identification of the cytochrome P450 isoforms involved

This study aims to characterize interindividual variability and individual CYP enzymes involved in the in vitro metabolism of the carbamate insecticide carbosulfan. Microsomes from ten human livers (HLM) were used to characterize the interindividual variability in carbosulfan activation. Altogether eight phase I metabolites were analyzed by LC–MS. The primary metabolic pathways were detoxification by the initial oxidation of sulfur to carbosulfan sulfinamide (‘sulfur oxidation pathway’) and activation via cleavage of the nitrogen sulfur bond (N–S) to give carbofuran and dibutylamine (‘carbofuran pathway’). Differences between maximum and minimum carbosulfan activation values with HLM indicated nearly 5.9-, 7.0, and 6.6-fold variability in the k_m, V_max and CL_int values, respectively. CYP3A5 and CYP2B6 had the greatest efficiency to form carbosulfan sulfinamide, while CYP3A4 and CYP3A5 were the most efficient in the generation of the carbofuran metabolic pathway. Based on average abundances of CYP enzymes in human liver, CYP3A4 contributed to 98% of carbosulfan activation, while CYP3A4 and CYP2B6 contributed 57 and 37% to detoxification, respectively. Significant correlations between carbosulfan activation and CYP marker activities were seen with CYP3A4 (omeprazole sulfoxidation), CYP2C19 (omeprazole 5-hydroxylation) and CYP3A4 (midazolam 1′-hydroxylation), displaying r² = 0.96, 0.87 and 0.82, respectively. Activation and detoxification pathways were inhibited by ketoconazole, a specific CYP3A4 inhibitor, by 90–97% and 47–94%, respectively. Carbosulfan inhibited relatively potently CYP3A4 and moderately CYP1A1/2 and CYP2C19 in pooled HLM. These results suggest that the carbosulfan activation pathway is more important than the detoxification pathway, and that carbosulfan activation is predominantly catalyzed in humans by CYP3A4.     

Single-enzyme kinetics with fluorogenic substrates: lessons learnt and future directions

Single-molecule fluorescence techniques have developed into powerful tools for studying the kinetics of biological reactions at the single-molecule level. Using fluorogenic substrates, enzymatic reactions can be observed in real-time with single-turnover resolution. The turnover sequence contains all kinetic information, giving access to reaction substeps and dynamic processes such as fluctuations in the reaction rate. Despite their clearly proven potential, the accuracy of current measurements is limited by the availability of substrates with 1:1 stoichiometry and the signal-to-noise ratio of the measurement. In this review we summarize the state-of-the-art and discuss these limitations using experiments performed with α-chymotrypsin as an example. We are further providing an overview of recent efforts aimed at the improvement of fluorogenic substrates and the development of new detection schemes. These detection schemes utilize nanophotonic structures such as zero mode waveguides or nanoantennas. Nanophotonic approaches reduce the size of the effective detection volume and are a powerful strategy to increase the signal-to-noise ratio. We believe that a combination of improved substrates and novel detection schemes will pave the way for performing accurate single-enzyme experiments in biologically relevant conditions.     

Activity traps and the corer: complementary methods for sampling aquatic invertebrates

We compared the picture of aquatic invertebrate fauna given by three types of sampling methods: the corer as well as bottom and mid-water activity traps. The corer mainly caught benthic animals (89%), the most numerous groups of which were chironomids and sphaerids. Nekton was more abundant in activity traps (77% in bottom and 63% in mid-water), the most numerous groups were Cladocera and water mites. The size distributions in catch of the corer and the activity traps differed but there was no difference between the two types of activity traps. In activity traps, individuals of the smallest size class were most abundant, wheras animals of the second smallest size class were most numerous in the corer. We suggest that activity traps and the corer could be used as complementary methods to sample aquatic invertebrate communities.     

Drosophila vitelline membrane genes contain a 114 base pair region of highly conserved coding sequence

Two Drosophila vitelline membrane (VM) genes located at polytene band positions 26A and 34C have been characterized at the nucleotide level. Sequence comparison of the two genes VM26A.1 and VM34C.1 has revealed a similar 114 base pair region centrally located in the coding regions of both genes. The conserved region has a 91% homology at the nucleic acid level and a 100% conservation at the amino acid level. This suggests a common evolutionary origin for these VM genes and indicates that a strong selective pressure exists to maintain a specific polypeptide sequence in a domain of the proteins.     

Fallen retention aspen trees on clear-cuts can be important habitats for red-listed polypores: a case study in Finland

Green-tree retention is a relatively new forestry application, which aims at decreasing the negative effects of clear-cut logging on forest biodiversity. In this study, the value of retained aspens in maintaining diverse assemblages of wood-decaying fungi (polypores; Basidiomycota) on clear-cuts was investigated, after the retention trees had died, fallen and started to decay. A total of 110 fallen aspen trunks were investigated on clear-cuts and within old-growth forests in eastern Finland, southern boreal zone; and 499 records of polypores belonging to 46 species were made. The intermediately decayed trunks on a clear-cut area hosted more species and more red-listed species than did trunks within forests. Most of the polypore species with more than two records were found in both habitats. These results suggest that many aspen-associated polypores are able to live and reproduce in sun-exposed habitats, if the quality and quantity of dead wood fulfill the species-specific requirements. This unexpected result, however, may be partly due to the exceptionally great abundance of aspen in the study area. Furthermore, in the long term, the local benefits of fallen retention trees can be limited, unless the local continuity of large aspens, both living and dead, is ensured.